Method for the synthesis of nucleotide or oligonucleotide phosphoramidites

ABSTRACT

This invention presents novel methods for recovery of phosphoramidites from the waste products of oligonucleotide synthesis. The methods include reacting a tribromophenoxydichlorophosphorane with a H-phosphonate in the presence of an amine.

FIELD OF THE INVENTION

This invention is directed to novel methods for the preparation ofphosphoramidites and oligophosphoramidites. The methods are useful,inter alia, for the preparation of phosphoramidites which are useful, inturn, in the synthesis of oligonucleotide diagnostic reagents, researchreagents and therapeutics agents.

BACKGROUND OF THE INVENTION

It is well known that most of the bodily states in mammals, includingmost disease states, are affected by proteins. Such proteins, eitheracting directly or through their enzymatic functions, contribute inmajor proportion to many diseases in animals and man. Classicaltherapeutics has generally focused on interactions with such proteins inefforts to moderate their disease causing or disease potentiatingfunctions. Recently, however, attempts have been made to moderate theactual production of such proteins by interactions with molecules thatdirect their synthesis, such as intracellular RNA. By interfering withthe production of proteins, it has been hoped to affect therapeuticresults with maximum effect and minimal side effects. It is the generalobject of such therapeutic approaches to interfere with or otherwisemodulate gene expression leading to undesired protein formation.

One method for inhibiting specific gene expression is the use ofoligonucleotides and oligonucleotide analogs as "antisense" agents. Theoligonucleotides or oligonucleotide analogs complimentary to a specific,target, messenger RNA (mRNA) sequence are used. Antisense methodology isoften directed to the complementary hybridization of relatively shortoligonucleotides and oligonucleotide analogs to single-stranded mRNA orsingle-stranded DNA such that the normal, essential functions of theseintracellular nucleic acids are disrupted. Hybridization is the sequencespecific hydrogen bonding of oligonucleotides or oligonucleotide analogsto Watson-Crick base pairs of RNA or single-stranded DNA. Such basepairs are said to be complementary to one another.

Prior attempts at antisense therapy have provided oligonucleotides oroligonucleotide analogs that are designed to bind in a specific fashionto a specific mRNA by hybridization (i.e., oligonucleotides that arespecifically hybridizable with a target mRNA). Such oligonucleotides andoligonucleotide analogs are intended to inhibit the activity of theselected mRNA by any of a number of mechanisms, i.e., to interfere withtranslation reactions by which proteins coded by the mRNA are produced.The inhibition of the formation of the specific proteins that are codedfor by the mRNA sequences interfered with have been hoped to lead totherapeutic benefits; however there are still problems to be solved. Seegenerally, Cook, P. D. Anti-Cancer Drug Design 1991, 6,585; Cook, P. D.Medicinal Chemistry Strategies for Antisense Research, in AntisenseResearch & Applications, Crooke, et al., CRC Press, Inc.; Boca Raton,Fla., 1993; Uhlmann, et al., A. Chem. Rev. 1990, 90, 543.

Oligonucleotides and oligonucleotide analogs are now accepted astherapeutic agents holding great promise for therapeutics anddiagnostics methods. But applications of oligonucleotides andoligonucleotide analogs as antisense agents for therapeutic purposes,diagnostic purposes, and research reagents often require that theoligonucleotides or oligonucleotide analogs be synthesized in largequantities, be transported across cell membranes or taken up by cells,appropriately hybridize to targeted RNA or DNA, and subsequentlyterminate or disrupt nucleic acid function. These critical functionsdepend on the initial stability of oligonucleotides and oligonucleotideanalogs toward nuclease degradation.

A serious deficiency of unmodified oligonucleotides for these purposes,particularly antisense therapeutics, is the enzymatic degradation of theadministered oligonucleotides by a variety of intracellular andextracellular ubiquitous nucleolytic enzymes.

A number of chemical modifications have been introduced into antisenseagents (i.e., oligonucleotides and oligonucleotide analogs) to increasetheir therapeutic activity. Such modifications are designed to increasecell penetration of the antisense agents, to stabilize the antisenseagents from nucleases and other enzymes that degrade or interfere withtheir structure or activity in the body, to enhance the antisenseagents' binding to targeted RNA, to provide a mode of disruption(terminating event) once the antisense agents are sequence-specificallybound to targeted RNA, and to improve the antisense agents'pharmacokinetic and pharmacodynamic properties. It is unlikely thatunmodified, "wild type," oligonucleotides will be useful therapeuticagents because they are rapidly degraded by nucleases.

Potential applications of these oligonucleotides and their modifiedderivatives as drugs have created new challenges in the large-scalesynthesis of these compounds.

The solid phase synthesis of oligonucleotides is inherently wasteful inthat more than one equivalent of nucleosidic phosphoramidite synthons isused presumably to drive the reaction to completion. Given the vastamounts of oligonucleotide syntheses performed for research use and forlarge scale manufacture pursuant to clinical trials, the waste ofexpensive nucleoside phosphoramidites is a significant economic andecological problem. This problem becomes more acute if one has tosynthesize the monomer through a multistep synthesis before reaching thephosphoramidite stage, as is the case where modified sugar or nucleobasecontaining synthons are used in oligonucleotide therapeutic agents.

Consequently, there remains a need in the art for synthetic methodswhich do not require the sacrifice of large amount of phosphoramiditereagent. The present invention addresses these, as well as other needs.

SUMMARY OF THE INVENTION

The present invention is directed to novel methods for the recovery ofphosphoramidites from waste products of traditional phosphoramiditesynthesis. In preferred embodiments, methods are provided for thepreparation of phosphoramidites comprising the steps of:

reacting a compound of formula: ##STR1## wherein: Z is aninternucleoside linkage;

R₁ is a hydroxyl protecting group;

R₂ is H, halogen, OH, O-alkyl, O-alkylamino, O-alkylalkoxy, a polyetherof formula (O-alkyl)_(m) where m is 1 to about 10, or a protectedhydroxyl group;

R₃ is a phosphoryl protecting group;

B is a nucleobase; and

n is 0 to about 100;

with a compound of formula: ##STR2## wherein: X₁ is Br or Cl;

X₂ and X₃ are each, independently, H, Br or Cl;

x and y are each, independently, 2 or 3, and the sum of x and y is 5;and

contacting the product of the reaction with a compound of formula HN(Q)₂to yield a phosphoramidite of formula: ##STR3##

In some preferred embodiments Q is alkyl, preferably isopropyl.

R₂ is preferably H, a protected hydroxyl group, O-alkyl orO-alkylalkoxy. In more preferred embodiments R₂ is methoxyethoxy.

In some preferred embodiments R₃ is cyanoethyl, 4-cyano-2-butenyl, ordiphenylmethylsilylethyl. Preferably, x is 3 and y is 2.

In prefered embodiments the reaction is performed in a solvent, which ispreferably acetonitrile.

In some preferred embodiments the nucleobase is adenine, guanine,cytosine, thymine, uracil, 5-methyl cytosine or a protected derivativethereof.

In further preferred embodiments Z is a phosphodiester linkage, aphosphorothioate linkage, a phosphorodithioate linkage; or a phosphonatelinkage. In particularly preferred embodiments Z is a phosphodiesterlinkage or a phosphorothioate linkage.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention presents novel methods for the preparation ofphosphoramidites. The methods are useful for the recovery of a widevariety of species produced as waste products in oligonucleotidesynthesis. In preferred embodiments, methods are provided for thepreparation of phosphoramidites of formula: ##STR4## wherein Z is aninternucleoside linkage; R₁ is a hydroxyl protecting group; R₂ is H,halogen, OH, O-alkyl, O-alkylamino, O-alkylalkoxy, a polyether offormula (O-alkyl)_(m) where m is 1 to about 10, or a protected hydroxylgroup; R₃ is a phosphoryl protecting group; B is a nucleobase; n is 0 toabout 100, and each Q is independently alkyl having from 1 to 15carbons, aryl having from 6 to 14 carbons, or (Q)₂ together with thephosphoamidite nitrogen can form a heterocyclic ring having from 2 to 10carbons. The methods comprise the steps of reacting a compound ofFormula I or II: ##STR5## with a halophenoxychlorophosphorane of formulaIII: ##STR6## wherein: X₁ is Br or Cl;

X₂ and X₃ are, independently, H, Br or Cl;

x and y are, independently, 2 or 3, with the sum of x and y being 5; andcontacting the product of the reaction with a compound of formulaHN(Q)₂.

Compounds of Formula I or II can derive from the waste excessphosphoramidite reagents used in traditional oligonucleotide synthesis,as described in, for example, Oligonucleotides and Analogues: APractical Approach, Eckstein, F., Ed., IRL Press, Oxford, U.K. 1991,which is incorporated herein by reference in its entirety. In a typicaloligonucleoide synthetic regime, the free 5-hydroxyl of the growingoligonucleotide chain is reacted with a several-fold molar excess ofnucleoside N,N-dialkylphosphoramidite in the presence of excesstetrazole. It is believed that the tetrazole catalyst first displacesthe secondary amino group of the phosphoramidite to form a tetrazolideaddduct. The tetrazolide then reacts with the free 5'-hydroxyl of thegrowing chain to form a phosphite, which is subsequently transformedinto the desired linkage by, for example, oxidation. The excess (i.e.,unreacted) terazolide adduct is washed from the reaction chamber, andsubsequently reacts with ambient water to form an H-phosphonate speciesrepresented by Formula I, which exists in equilibrium with its tautomer,represented by Formula II. Thus, in one aspect, the present inventionprovides a convenient method of recovering phosphoramidite synthons fromwaste products of oligonucleotide synthesis.

The methods of the present invention provide significant economic andecological benefits in oligonucleotide synthesis. While not wishing tobe bound by a particular theory, it is believed that the phosphonatespecies (Formula I) and its phosphite tautomer (Formula II) exist inequilibrium in solution, with the phosphonate species being heavilyfavored. The halophenoxychlorophosphorane, preferablytris(2,4,6-tribromophenoxy)dichlorophosphorane or bis2,4,6-tribromophenoxy!trichlorophosphorane, is believed topreferentially react with the phosphite species to form a halide adduct,which then reacts with a secondary amine to form the phosphoramiditeproduct. Accordingly, the methods of the present invention can beconveniently perfomed in a single reaction container, or in stages.

The synthons of Formula I or II are derived from any of the wide varietyof phosphoramidite species capable of being used in phosphoramiditeologomer synthesis. Accordingly, the synthons can be monomeric, dimeric,or higher order synthons (i.e., oligophosphoramidites), and can compriseany of the wide variety of internucleoside linkages, sugars,nucleobases, and modified derivatives thereof known in the art.

Examples of internucleoside linkages which can be present in synthons ofFormula I or II include phosphodiester, phosphorothioate,phosphorodithioate, and phosphonate linkages. Further representativeinternucleotide linkages include amide or substituted amide linkages,such as those described in Waldner et al., Synlett. 1, 57-61 (1994), DeMesmaeker et al., Synlett. 10, 733-736 (1993), Lebreton et al., Synlett.2, 137-140 (1994), De Mesmaeker et al., Bioorg. Medic. Chem. Lett. 4,395-398 (1994), De Mesmaeker et al., Bioorg. Medic. Chem. Lett. 4,873-878 (1994), Lebreton et al., Tet. Letters 34, 6383-6386 (1993),Lebreton et al., Tet. Letters 35, 5225-5228 (1994), Waldner et al.,Bioorg. Medic. Chem. Lett. 4, 405-408 (1994), and linkgaes described inU.S. Pat. No. 5,489,677, U.S. Ser. No. 08/317,289, filed Oct. 3, 1994,U.S. Ser. No. 08/395,168, filed Feb. 27, 1995.

In the context of the present invention, the term "oligonucleotide"refers to a plurality of joined nucleotide units formed in a specificsequence. The term nucleotide has its accustomed meaning as thephosphoryl ester of a nucleoside. The term "nucleoside" also has itsaccustomed meaning as a pentofuranosyl sugar which is bound to anucleosidic base (i.e, a nitrogenous heterocyclic base or "nucleobase").

It will be appreciated that the methods of the present invention can beused for the synthesis of phosphoramidites having both naturallyoccurring and non-naturally occurring constituent sugars,internucleoside linkages and/or nucleobases (i.e., nucleosidic bases).Non-naturally occurring sugars, internucleoside linkages and nucleobasesare typically structurally distinguishable from, yet functionallyinterchangeable with, naturally occurring sugars (e.g. ribose anddeoxyribose), internucleoside linkages (i.e. phosphodiester linkages),and nucleosidic bases (e.g., adenine, guanine, cytosine, thymine). Thus,non-naturally occurring moieties include all such structures which mimicthe structure and/or function of naturally occurring moiety, and whichaid in the binding of the oligonucleotide analog to a target, orotherwise advantageously contribute to the properties of thephosphorothioate oligomer.

Representative examples of non-naturally occurring sugars include sugarshaving any of a variety of substituents attached to their 2'-positions.These include, for example, halides, O-alkyl, O-alkylamino,O-alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, O-alkylimidazole, and polyethers of the formula (O-alkyl)_(m), where m is 1 toabout 10. Preferred among these polyethers are linear and cyclicpolyethylene glycols (PEGs), and (PEG)-containing groups, such as crownethers and those which are disclosed by Ouchi, et al., Drug Design andDiscovery 1992, 9, 93, Ravasio, et al., J. Org. Chem. 1991, 56, 4329,and Delgardo et. al., Critical Reviews in Therapeutic Drug CarrierSystems 1992, 9, 249. Further sugar modifications are disclosed in Cook,P. D., supra. Fluoro, O-alkyl, O-alkylamino, O-alkyl imidazole,O-alkylaminoalkyl, and alkyl amino substitution is described in U.S.patent application Ser. No. 08/398,901, filed Mar. 6, 1995, entitledOligomeric Compounds having Pyrimidine Nucleotide(s) with 2' and 5'Substitutions, the disclosure of which is hereby incorporated byreference.

Sugars having O-substitutions on the ribosyl ring are also amenable tothe present invention. Representative substitutions for ring O includeS, CH₂, CHF, and CF₂, see, e.g., Secrist, et al., Abstract 21, Program &Abstracts, Tenth International Roundtable, Nucleosides, Nucleotides andtheir Biological Applications, Park City, Utah, Sep. 16-20, 1992.

Representative nucleobases suitable for use in the methods of theinvention include adenine, guanine, cytosine, uridine, and thymine, aswell as other non-naturally occurring and natural nucleobases such asxanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 5-halo uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil, 8-halo,oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adeninesand guanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, 7-methylguanine. Further naturally and non-naturallyoccurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808(Merigan, et al.), by Sanghvi, Y., in chapter 15 of Antisense Researchand Application, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, inEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613-722 (see especially pages 622 and 623), in the Concise Encyclopediaof Polymer Science and Engineering, J. I. Kroschwitz Ed., John Wiley &Sons, 1990, pages 858-859, and in Cook, P. D., Anti-Cancer Drug Design,1991, 6, 585-607. The disclosures of each of the foregoing isincorporated by reference in their entirety. The terms "nucleosidicbase" and "nucleobase" are further intended to include heterocycliccompounds that can serve as nucleosidic bases, including certain`universal bases` that are not nucleosidic bases in the most classicalsense, but function similarly to nucleosidic bases. One representativeexample of such a universal base is 3-nitropyrrole.

In some preferred embodiments of the invention R₁ is a hydroxylprotecting group. A wide variety of hydroxyl protecting groups can beemployed in the methods of the invention. Preferably, the protectinggroup is stable under basic conditions but can be removed under acidicconditions. Representative hydroxyl protecting groups are disclosed byBeaucage, et al., Tetrahedron 1992, 48, 2223-2311, and also in e.g.,Green and Wuts, Protective Groups in Organic Synthesis, 2d edition, JohnWiley & Sons, New York, 1991 at Chapter 2. Preferred protecting groupsused for R₁, include dimethoxytrityl (DMT), monomethoxytrityl,9-phenylxanthen-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthen-9-yl (Mox).The R₁, group can be removed from oligomeric compounds of the inventionby techniques well known in the art to form the free hydroxyl. Forexample, dimethoxytrityl protecting groups can be removed by proticacids such as formic acid, dichloroacetic acid, trichloroacetic acid,p-toluene sulphonic acid or with Lewis acids such as for example zincbromide.

In some preferred embodiments of the invention R₃ is a phosphorylprotecting group. The phosphoryl protecting group is attached to thephosphorus-bound oxygen, and serves to protect the phosphorus duringoligonucleotide synthesis. See Oligonucleotides and Analogues: APractical Approach, supra. One representative phosphoryl protectinggroup is the cyanoethyl group. Other representative phosphorylprotecting groups include 4-cyano-2-butenyl groups, methyl groups, anddiphenylmethylsilylethyl (DPSE) groups.

In general, protecting groups are used in the oligonucleotide syntheticmethods of the invention for protection of several different types offunctionality. In general, protecting groups render chemicalfunctionality inert to specific reaction conditions and can be appendedto and removed from such functionality in a molecule withoutsubstantially damaging the remainder of the molecule. Representativeprotecting groups useful to protect nucleotides during phosphorothioatesynthesis include base labile protecting groups and acid labileprotecting groups. Base labile protecting groups are used to protect theexocyclic amino groups of the heterocyclic nucleobases. This type ofprotection is generally achieved by acylation. Two commonly usedacylating groups are benzoylchloride and isobutyrylchloride. Theseprotecting groups are stable to the reaction conditions used in themethods of the invention, and during oligonucleotide synthesis, and arecleaved at approximately equal rates during the base treatment at theend of oligonuclotide synthesis. The second type of protection, alsoused in the synthetic methods of the invention, is an acid labileprotecting group, which is used to protect the nucleotide 5'-hydroxylduring synthesis.

Tris 2,4,6-tribromophenoxy!dichlorophosphorane or bis2,4,6-tribromophenoxy!trichlorophosphorane can be synthesized accordingto the method of Hotoda et al., Tetrahedron Letters 1987 28 (15)1681-1684.

In the methods of the present invention, the product of the reactionbetween the compound of Formula I or II and thetribromophenoxychlorophosphorane reacts with a secondary amine to formthe phosphoramidite product. The substituents of the secondary amine canbe chosen from among the many species that are know to function asphosphoramidite nitrogen substituents. Representative examples includelower alkyl gourps, aryl groups, and cyclic structure such as where thephosphoramidite nitrogen forms part of a N-morpholine ring system. Inparticularly preferred embodiments the substituents are lower alkylgroups, especially isopropyl groups. Other examples of suitable aminesas are described in various United States patents, principally those toM. Caruthers and associates. These include U.S. Pat. Nos. 4,668,777;4,458,066; 4,415,732; and 4,500,707; all of which are hereinincorporated by reference.

As used herein, the term "alkyl" includes but is not limited to straightchain, branch chain, and alicyclic hydrocarbon groups. Alkyl groups ofthe present invention may be substituted. Representative alkylsubstituents are disclosed in U.S. Pat. No. 5,212,295, at column 12,lines 41-50.

As used herein, the term "aralkyl" denotes alkyl groups which bear arylgroups, for example, benzyl groups. The term "alkaryl" denotes arylgroups which bear alkyl groups, for example, methylphenyl groups. "Aryl"groups are aromatic cyclic compounds including but not limited tophenyl, naphthyl, anthracyl, phenanthryl, pyrenyl, and xylyl.

In some preferred embodiments of the invention amino groups are appendedto alkyl or other groups, such as, for example, 2'-alkoxy groups (e.g.,where R₂ is alkoxy). Such amino groups are also commonly present innaturally occurring and non-naturally occurring nucleobases. It isgenerally preferred that these amino groups be in protected form duringthe synthesis of oligomeric compounds of the invention. Representativeamino protecting groups suitable for these purposes are discussed inChapter 7 of Greene and Wuts, supra. Generally, as used herein, the term"nucleobase" includes protected derivatives thereof.

Oligomer phosphoramidites produced by the methods of the invention willpreferably comprise from about 1 to about 100 monomer subunits. It ismore preferred that such compounds comprise from about 1 to about 30monomer subunits, with 1 to 10 monomer subunits being more preferred,and 1 to 5 monomer subunits being particularly preferred.

Additional advantages and novel features of this invention will becomeapparent to those skilled in the art upon examination of the examplesthereof provided below, which should not be construed as limiting theappended claims.

EXAMPLE 1 Preparation of Tris(2,4,6-tribromophenoxy)dichlorophosphorane

This compound was synthesized according the procedure of Hotoda, H. etal., Tetrahedron Letters, 1987, Vol. 28, 1681-1684.

EXAMPLE 2 Preparation of 5'-O-(4,4'dimethoxytrityl)thymidine-3'-O-(2-diphenylmethylsilylethyl N,N-diisopropylphosphoramidite)

A 250 mL two necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. Allglassware are dried at 120° C. for 1 hour. The flask is charged with5'-O-(4,4'dimethoxytrityl)thymidine-3'-O-(2-diphenylmethylsilylethylphosphonate)(0.015 mole) and diisopropylamine (0.12 mole). Anhydrous acetonitrile(200 mL) is added. To this stirred mixture under argon at roomtemperature is added tris(2,4,6-tribromophenoxy)dichlorophosphorane(0.225 mole). After stirring for three hours, the reaction mixture isfiltered, and concentrated under reduced pressure. The crude product ispurified by flash chromatography using silica gel to afford the desiredproduct. Triethylamine (1%) is used throughout the purification. ³¹ PNMR (CDCl₃) 145.5, 146.1

EXAMPLE 3 Preparation of N²-Isobutyryl-5'-O-(4,4'dimethoxytrityl)-2'-deoxyguanosine-3'-O-(2-diphenylmethylsilylethylN,N-diisopropylphosphoramidite)

A 250 mL two necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. Allglassware are dried at 120° C. for 1 hour. The flask is charged with N²-Isobutyryl-5'-O-(4,4'dimethoxytrityl)-2'-deoxyguanosine-3'-O-(2-diphenylmethylsilylethylphosphonate)(0.015 mole) and diisopropylamine (0.12 mole). Anhydrous acetonitrile(200 mL) is added. To this stirred mixture under argon at roomtemperature is added tris(2,4,6-tribromophenoxy)dichlorophosphorane(0.225 mole). After stirring for three hours, the reaction mixture isfiltered, and concentrated under reduced pressure. The crude product ispurified by flash chromatography using silica gel to afford the desiredproduct. Triethylamine (1%) is used throughout the purification.

EXAMPLE 4 Preparation of N⁶-Benzoyl-5'-O-(4,4'dimethoxytrityl)-2'-deoxyadenosine-3'-O-(2-diphenylmethylsilylethylN,N-diisopropylphosphoramidite)

A 250 mL two necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. Allglassware are dried at 120° C. for 1 hour. The flask is charged with N⁶-Benzoyl-5'-O-(4,4'dimethoxytrityl)-2'-deoxyadenosine-3'-O-(2-diphenylmethylsilylethylphosphonate)(0.015 mole) and diisopropylamine (0.12 mole). Anhydrous acetonitrile(200 mL) is added. To this stirred mixture under argon at roomtemperature is added tris(2,4,6-tribromophenoxy)dichlorophosphorane(0.225 mole). After stirring for three hours, the reaction mixture isfiltered, and concentrated under reduced pressure. The crude product ispurified by flash chromatography using silica gel to afford the desiredproduct. Triethylamine (1%) is used throughout the purification.

EXAMPLE 5 Preparation of N⁴-Benzoyl-5'-O-(4,4'dimethoxytrityl)-2'-deoxycytidine-3'-O-(2-diphenylmethylsilylethylN,N-diisopropylphosphoramidite)

A 250 mL two necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. Allglassware are dried at 120° C. for 1 hour. The flask is charged with N⁴-Benzoyl-5'-O-(4,4'dimethoxytrityl)-2'-deoxycytidine-3'-O-(2-diphenylmethylsilylethylphosphonate)(0.015 mole) and diisopropylamine (0.12 mole). Anhydrous acetonitrile(200 mL) is added. To this stirred mixture under argon at roomtemperature is added tris(2,4,6-tribromophenoxy)dichlorophosphorane(0.225 mole). After stirring for three hours, the reaction mixture isfiltered, and concentrated under reduced pressure. The crude product ispurified by flash chromatography using silica gel to afford the desiredproduct. Triethylamine (1%) is used throughout the purification.

EXAMPLE 62-Diphenylmethylsilylethyl-5'-(O-4,4'-dimethoxytrityl)thymidinyl-thymidinedimer amidite

A 250 mL two necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. Allglassware are dried at 120° C. for 1 hour. The flask is charged with2-diphenylmethylsilylethyl-5'-(O-4,4'-dimethoxytrityl)thymidinyl-thymidinedimer phosphonate (0.015 mole) and diisopropylamine (0.12 mole).Anhydrous acetonitrile (200 mL) is added. To this stirred mixture underargon at room temperature is addedtris(2,4,6-tribromophenoxy)dichlorophosphorane (0.225 mole). Afterstirring for three hours, the reaction mixture is filtered, andconcentrated under reduced pressure. The crude product is purified byflash chromatography using silica gel to afford the desired product.Triethylamine (1%) is used throughout the purification.

EXAMPLE 72-Diphenylmethylsilylethyl-5'-(O-4,4'-dimethoxytrityl)-N4-benzoyl-2'-deoxycytidinyl-thymidinedimer amidite

A 250 mL two necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. Allglassware are dried at 120° C. for 1 hour. The flask is charged with2-diphenylmethylsilylethyl-5'-(O-4,4'-dimethoxytrityl)-N4-benzoyl-2'-deoxycytidinyl-thymidinedimer phosphonate (0.015 mole) and diisopropylamine (0.12 mole).Anhydrous acetonitrile (200 mL) is added. To this stirred mixture underargon at room temperature is addedtris(2,4,6-tribromophenoxy)dichlorophosphorane (0.225 mole). Afterstirring for three hours, the reaction mixture is filtered, andconcentrated under reduced pressure. The crude product is purified byflash chromatography using silica gel to afford the desired product.Triethylamine (1%) is used throughout the purification.

EXAMPLE 82-Diphenylmethylsilylethyl-5'-(O-4,4'-dimethoxytrityl)-N2-isobutyryl-2'-deoxyguanosinyl-thymidinedimer amidite

A 250 mL two necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. Allglassware are dried at 120° C. for 1 hour. The flask is charged with2-diphenylmethylsilylethyl-5'-(O-4,4'-dimethoxytrityl)-N2-isobutyryl-2'-deoxyguanosinyl-thymidinedimer phosphonate (0.015 mole) and diisopropylamine (0.12 mole).Anhydrous acetonitrile (200 mL) is added. To this stirred mixture underargon at room temperature is addedtris(2,4,6-tribromophenoxy)dichlorophosphorane (0.225 mole). Afterstirring for three hours, the reaction mixture is filtered, andconcentrated under reduced pressure. The crude product is purified byflash chromatography using silica gel to afford the desired product.Triethylamine (1%) is used throughout the purification.

EXAMPLE 92-Diphenylmethylsilylethyl-5'-(O-4,4'-dimethoxytrityl)-N6-benzoyl-2'-deoxyadenosinyl-thymidinedimer amidite

A 250 mL two necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. Allglassware are dried at 120° C. for 1 hour. The flask is charged with2-diphenylmethylsilylethyl-5'-(O-4,4'-dimethoxytrityl)-N6-benzoyl-2'-deoxyadenosinyl-thymidinedimer phosphonate (0.015 mole) and diisopropylamine (0.12 mole).Anhydrous acetonitrile (200 mL) is added. To this stirred mixture underargon at room temperature is addedtris(2,4,6-tribromophenoxy)dichlorophosphorane (0.225 mole). Afterstirring for three hours, the reaction mixture is filtered, andconcentrated under reduced pressure. The crude product is purified byflash chromatography using silica gel to afford the desired product.Triethylamine (1%) is used throughout the purification.

EXAMPLE 102-Diphenylmethylsilylethyl-5'-(O-4,4'-dimethoxytrityl)-N2-isobutyryl-2'-deoxyguanosinyl-N6-benzoyl-2'-deoxyadenosinyldimer amidite

A 250 mL two necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. Allglassware are dried at 120° C. for 1 hour. The flask is charged with2-diphenylmethylsilylethyl-5'-(O-4,4'-dimethoxytrityl)-N2-isobutyryl-2'-deoxyguanosinyl-N6-benzoyl-2'-deoxyadenosinyldimer phosphonate (0.015 mole) and diisopropylamine (0.12 mole).Anhydrous acetonitrile (200 mL) is added. To this stirred mixture underargon at room temperature is addedtris(2,4,6-tribromophenoxy)dichlorophosphorane (0.225 mole). Afterstirring for three hours, the reaction mixture is filtered, andconcentrated under reduced pressure. The crude product is purified byflash chromatography using silica gel to afford the desired product.Triethylamine (1%) is used throughout the purification.

EXAMPLE 11 Preparation of5'-O-(4,4'dimethoxytrityl)uridine-2'-O-methoxyethyl-3'-O-(2-diphenylmethylsilylethylN,N-diisopropylphosphoramidite)

A 250 mL two necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. Allglassware are dried at 120° C. for 1 hour. The flask is charged with5'-O-(4,4'dimethoxytrityl)uridine-2'-O-methoxyethyl-3'-O-(2-diphenylmethylsilylethylphosphonate)(0.015 mole) and diisopropylamine (0.12 mole). Anhydrous acetonitrile(200 mL) is added. To this stirred mixture under argon at roomtemperature is added tris(2,4,6-tribromophenoxy)dichlorophosphorane(0.225 mole). After stirring for three hours, the reaction mixture isfiltered, and concentrated under reduced pressure. The crude product ispurified by flash chromatography using silica gel to afford the desiredproduct. Triethylamine (1%) is used throughout the purification. ³¹ PNMR (CDCl₃) 145.5, 146.1

EXAMPLE 12

Preparation of N²-Isobutyryl-5'-O-(4,4'dimethoxytrityl)-2'-O-methoxyethylguanosine-3'-O-(2-diphenylmethylsilylethylN,N-diisopropylphosphoramidite)

A 250 mL two necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. Allglassware are dried at 120° C. for 1 hour. The flask is charged with N²-Isobutyryl-5'-O-(4,4'dimethoxytrityl)-2'-O-methoxyethylguanosine-3'-O-(2-diphenylmethylsilylethylphosphonate)(0.015 mole) and diisopropylamine (0.12 mole). Anhydrous acetonitrile(200 mL) is added. To this stirred mixture under argon at roomtemperature is added tris(2,4,6-tribromophenoxy)dichlorophosphorane(0.225 mole). After stirring for three hours, the reaction mixture isfiltered, and concentrated under reduced pressure. The crude product ispurified by flash chromatography using silica gel to afford the desiredproduct. Triethylamine (1%) is used throughout the purification.

EXAMPLE 13 Preparation of N⁶-Benzoyl-5'-O-(4,4'dimethoxytrityl)-2'-O-methoxyethyladenosine-3'-O-(2-diphenylmethylsilylethylN,N-diisopropylphosphoramidite)

A 250 mL two necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. Allglassware are dried at 120° C. for 1 hour. The flask is charged with N⁶-Benzoyl-5'-O-(4,4'dimethoxytrityl)-2'-O-methoxyethyladenosine-3'-O-(2-diphenylmethylsilylethylphosphonate)(0.015 mole) and diisopropylamine (0.12 mole). Anhydrous acetonitrile(200 mL) is added. To this stirred mixture under argon at roomtemperature is added tris(2,4,6-tribromophenoxy)dichlorophosphorane(0.225 mole). After stirring for three hours, the reaction mixture isfiltered, and concentrated under reduced pressure. The crude product ispurified by flash chromatography using silica gel to afford the desiredproduct. Triethylamine (1%) is used throughout the purification.

EXAMPLE 14 Preparation of N⁴-Benzoyl-5'-O-(4,4'dimethoxytrityl)-2'-O-methoxyethylcytidine-3'-O-(2-diphenylmethylsilylethylN,N-diisopropylphosphoramidite)

A 250 mL two necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. Allglassware are dried at 120° C. for 1 hour. The flask is charged with N⁴-Benzoyl-5'-O-(4,4'dimethoxytrityl)-2'-O-methoxyethylcytidine-3'-O-(2-diphenylmethylsilylethylphosphonate)(0.015 mole) and diisopropylamine (0.12 mole). Anhydrous acetonitrile(200 mL) is added. To this stirred mixture under argon at roomtemperature is added tris(2,4,6-tribromophenoxy)dichlorophosphorane(0.225 mole). After stirring for three hours, the reaction mixture isfiltered, and concentrated under reduced pressure. The crude product ispurified by flash chromatography using silica gel to afford the desiredproduct. Triethylamine (1%) is used throughout the purification.

It is intended that each of the patents, applications, printedpublications, and other published documents mentioned or referred to inthis specification be herein incorporated by reference in theirentirety.

As those skilled in the art will appreciate, numerous changes andmodifications may be made to the preferred embodiments of the inventionwithout departing from the spirit of the invention. It is intended thatall such variations fall within the scope of the invention.

What is claimed is:
 1. A method for the preparation of a phosphoramiditecomprising the steps of:reacting a compound of formula:formula: ##STR7##wherein: Z is an intersugar linkage; R₁ is a hydroxyl protecting group;R₂ is H, OH, O-alkyl, O-alkylamino, O-alkylalkoxy, a polyether offormula (O-alkyl)_(m) where m is 1 to about 10, or a protected hydroxylgroup; R₃ is a phosphoryl protecting group; B is a nucleobase; and n is0 to about 100;with a compound of formula: ##STR8## wherein: X₁ is Br orCl; X₂ and X₃ are, independently, H, Br or Cl; x and y are each,independently, 2 or 3, and the sum of x and y is 5; and contacting theproduct of the reaction with a compound of formula HN(Q)₂ to yield aphosphoramidite of formula: ##STR9## wherein each Q is independentlyalkyl having from 1 to 15 carbons, aryl having from 6 to 14 carbons orcan form a heterocyclic ring having from 2 to 10 carbons with thenitrogen of the phosphoramidite.
 2. The method of claim 1 wherein Q isdiisopropyl.
 3. The method of claim 1 wherein R₂ is H.
 4. The method ofclaim 1 wherein R₂ is a protected hydroxyl group.
 5. The method of claim1 wherein R₂ is O-alkyl or O-alkylalkoxy.
 6. The method of claim 1wherein R₂ is O-alkylalkoxy.
 7. The method of claim 1 wherein R₂ is2-methoxyethoxy.
 8. The method of claim 1 wherein R₃ is cyanoethyl,4-cyano-2-butenyl, or diphenylmethylsilylethyl.
 9. The method of claim 1wherein x is 2 and y is
 3. 10. The method of claim 1 wherein x is 3 andy is
 2. 11. The method of claim 1 wherein the reaction is performed inthe presence of a solvent.
 12. The method of claim 11 wherein thereaction is performed in the presence of a solvent is acetonitrile. 13.The method of claims 1 wherein the nucleobase is adenine, guanine,cytosine, thymine, uracil, 5-methyl cytosine or a protected derivativethereof.
 14. The method of claim 1 wherein Z is a phosphodiesterlinkage, a phosphorothioate linkage, a phosphorodithioate linkage; or aphosphonate linkage.
 15. The method of claim 14 wherein Z is aphosphodiester linkage or a phosphorothioate linkage.
 16. The method ofclaim 1 wherein R₃ is cyanoethyl.
 17. The method of claim 1 wherein R₃is 4-cyano-2-butenyl.
 18. The method of claim 1 wherein X₁, X₂ and X₃are Br located at 2,4 and 6-positions of the phenyl ring.